Process for preparing an antipoliomyelitis vaccine



From German Patent 1,075,279 it is known to prepare a vaccine byinactivating viruses of foot and mouth disease, of the encephalomyelitisgroup, or myxo group by treating them with hydroxylamme.

Attempts to apply this hydroxylamine inactivation process topoliomyelitis viruses, which are enteroviruses in contradistinction tothe above-cited viruses I have heretofore been unsuccessful. In view ofthe strong pathogenicity of the poliomyelitis viruses, exactingregulations regarding the production and the testing of polioviruscontaining vaccines have been established. Thus, vaccines containinginactivated poliomyelitis viruses are required to be absolutely freefrom infectious viruses (apathogenicity), and it is required that thecourse of the virus inactivation be clearly controllable. This latterrequirement is not always met by the conventional inactivation methodswith formaldehyde and heat.

For producing poliomyelitis viruses for the preparation ofantipoliomyelitis vaccines, primary cell tissue cultures of monkeykidneys are used. In this process, impurities in the form of foreignviruses, which are called simian viruses or pick-up viruses, are oftenobserved.

The presence of such foreign viruses does not affect the preparation ofantipoliomyelitis vaccines, so long as the foreign viruses can beinactivated in the same manner with formaldehyde with at least the samespeed as the poliornyelitis viruses. But, there has been recentlydiscovered one simian virus which is not inactivated by formaldehyde;this virus is called simian virus 40, ab-

breviated SV40, or vacuolating virus.

No process has heretofore been known which permits the inactivation ofSV40 associated with poliomyelitis viruses during the preparation ofantipoliomyelitis vac cines without aifecting the poliomyelitis viruses.

Attempts have been made to inactivate SV40 with the aid of magnesiumchloride. According to said method, the magnesium chloride-containingsuspension is heated to 50f C. But despite the heating, this method doesnot produce the desired result, since the SV40 remains alive; on thecontrary, the antigenicity of the poliovirus is affected by the heating.

Now, we'have found a process for preparing an antipoliomyelitis vaccinewhich consists in adding hydroxylamine in a concentration from 0.01 to5.0 mols/liter, preferably 0.2 moi/liter, to a suspension of'poliomyelitis viruses which may be associated with SV40 or vacuo latingviruses, at a pH value ranging from 4.5 to 6.5, preferably 6.0, and at atemperature in the range from 4 to 40 C., preferably C., or at a pHvalue of 7.0 and at a temperature in the range from to C., preferably 37C. out after concentration of the suspension. The hydroxylamine isallowed to act on the virus suspension until the suspension is free frominfectious viruses. Then, un-

United States Patent 0 its original volume.

3,l97,1i72 Patented July 27, 1965 ice erably aluminum hydroxide and/ oraluminum phosphate, for increasing antibody formation, the suspension ofinactivated virus can be processed by conventional techniques into avaccine.

Thus, in the process of the present invention, the virus material issubjected to inactivation at either a neutral pH value and a temperaturein the range from 30 to 40 C. or at a pH value ranging from 4.5 to 6.5,preferably 6, and a temperature in the range from 4 to 40 C., preferably20 C., in order to neutralize any SV40 present.

In the process of the present invention, the period 0 time required forcomplete neutralization by the hydroxylamine of the infectiousness ofthe polioviruses can be easily determined. It is a characteristicfeature of the hydroxylamine inactivation of polioviruses according tothis invention that the course of the inactivation can be plotted as alinear function of time which can be extrapolated to a zeroconcentration of the infectious germs. This is not possible informaldehyde inactivation. The function of the inactivation to time isin that case not a straight line.

Furthermore, the process of the present invention offers particularadvantages in that the inactivation is completed Within a few hours andthe at the immunogenic effectiveness is not deteriorated by the process.In contrast thereto, formaldehyde inactivation under ordinary conditionsrequires 9-12 days and involves also a loss of antigenic activity causedby formaldehyde.

For interrupting the inactivation process, a carbonyl compound,preferably acetone, is advantageously added in one to ten times thestoichiometrical amount, calculated on hydroxylamine. The oxime thatforms during the reaction of the hydroxylamine with thecarbonyl-containing substance can be removed by dialysis. Ifcarbonylcontaining substances are used which yield water-insolubleoximes, for example, camphor or steroid ketones, the oximes can beremoved from the reaction solution by centrifugal action or byfiltration.

The virus suspension obtained in this manner. may be concentrated, ifdesired or required, and combined with adjuvants, preferably aluminumhydroxide and/or aluminum phosphate, in order to increase antibodyformation. It is also possible to concentrate the starting virussuspension prior to inactivation, for example, by ultrafiltration or bychromatography on a calcium phosphate column, and then to inactivatewith hydroxylamine the product concentrated, e.g., as in Example 3, toof The inactivated concentrated virus suspension may then be dilutedagain.

This method of operation according to the present invention saves work,material, and space. Because of If desired, this addition may be carriedreacted hydroxylamine is bound by means of a carbonylcontainingcompound, preferably acetone. If desired or required, the oxime compoundsimultaneously formed is removed by, for example, dialysis or, if theoxime is insoluble, by centrifugal action or by filtration, and finally,after having concentrated and/or added adjuvants, prefconcentration, alloperations can be performed with smaller volumes. In addition thereto,this procedure also brings about an additional dilution of the unreactedhydroxylamine and of its reaction product with the carbonyl-containingcompound added. Finally, it permits adjustment of the antigenic activityof the poliovaccines prepared to a determined value this assuring theproduction of-a Vaccine with constant antigenic activity,

The following examples illustrate the invention, but they are notintended to limit it thereto:

Example 1 (a) 50 cc. of a birnolar neutral solution of hydroxylamine areadded, at 20 C., to 450 cc. of a suspension of poliomyelitis virus typeI/Mahoney, having an infectivity of 'l0 lD /cc. and prepared in knownmanner, and the Whole is mixed thoroughly. Every 3 hours, samples of 5cc. are taken from the mixture and to each sample there is added 0.15cc. of acetone. These samples are then tested for infectivity in tissuecultures and the data obtained are plotted on a network of coordinatesversus time. The plotted values are all located on a straight line. Byextension of this line it is possible to determine the time at which thevirus suspension would be free from infectious viruses. Extrapolationshowed that the suspension would be free from infectious viruses after aperiod of 50 hours.

(b) 500 cc. of a bimolar neutral hydroxylamine solution are added at 20C. to 4500 cc. of the above starting material; after 50 hours, 150 cc.of acetone are added and the whole is mixed with 388 cc. of an Al(GH)suspension of 1% strength. 5538 cc. of a polio-vaccine type I/Mahoneyare obtained.

Test for antigenicity-The antigenic activity is determined by tests inchickens and in guinea pigs, as usual with poliomyelitis viruses. Forthus purpose, two vac cines are prepared starting from equal infectivitytiters by inactivation with formaldehyde and hydroxylamine,respectively. The specific antibody titer in the test animals is thendetermined in known manner with the following results:

Antibody titer in the serum Inactivation Agent In chickens In guineaFormaldehyde 1:40 1:128 Hydroxylamine 1:64 1:256

Example 2 4500 cc. each of poliovirus suspensions of the three typesI/Mahoney having an infectivity of 10"- II/M.E.F. 1 having aninfectivity of 10 III/Saukett having an infectivity of 10 compared withdata of a poliovaccine prepared with the same starting material butinactivated with formaldehyde.

Antibody titer in the serum in guinea pigs test Inactwatmg agent Type IType II Type HI Formaldehyde 1:128 1: 112 1: 64

Hydroxylamine 1:256 1:512 1: 161

Example 3 450 liters of the starting material used in Example 1 areconcentrated, Without loss of infectivity, by ultrafiltration to 4.5liters and treated as described in Example 1. After the addition ofacetone, the inactivated virus suspension is again diluted to 450 literswith a Hanks- TCM-soiution (75:25) and processed into a vaccine withAl(OH) Example 4 In a batch comprising 45 cc. of the poliovirussuspension prepared according to Example 1, the unreacted hydroxylamineis neutralized by the addition of 2.05 cc. of acetylacetone. The batchis then processed into a vaccine as described in Examples 1-3.

Example 5 90 cc. of a suspension of poliomyelitis viruses of type dII/MEJF. 1 having an infectivity titer of l0' iD /cc. are inactivated at20 C. by means of 10 cc. of a bimolar hydroxylamine solution. Unreactedhydroxylamine is neutralized by the addition of 4.70 cc. ofacetonylacetone. The batch is then processed into a vaccine as describedin Examples 1-3.

Example 6 cc. of a suspension of poliomyelitis viruses of type I/Mahoneyhaving an infectivity titer of IO ID /cc. are inactivated as describedin Example 5; unreacted hydroxylamine is neutralized by the addition of5.10 cc. of acetoacetic acid ethyl ester. The batch is then processedinto a vaccine as described in Examples 1-3.

Example 7 5 cc. of a bimolar neutral hydroxylamine solution are added at20 C. to 45 cc. of a suspension of poliomyeli tiS' viruses of the typeIII/Saukctt having an infectivity of 10 -ID cc. and prepared in knownmanner; the whole is well mixed and allowed to stand for 50 hours. thistime all poliomyelitis viruses are inactivated. Unreacted hydroxylamineis neutralized by the addition of 2.17 cc. of acetoacetic acid methylester. The batch is processed into a vaccine as described in Examples1-3.

Example 8 50 cc. of a bimolar hydroxylamine solution are added at 20 C.to 450 cc. of a suspension of poliomyelitis viruses type I/Mahoneyprepared in known manner and having an infectivity of l0' lD /cc. and anadditional SV40 infectivity determined on titration in tissue culturesof African green monkeys, Cercopithecus aetlziops; the whole is wellmixed and adjusted to a pH value of 6.0. After having allowed thehydroxylamine to act during 50 hours at 20 C., unreacted hydroxylamineis neutralized with 15 cc. of acetone. Thereafter, the virus suspensionis free from infectious viruses The antigenicity of the polioviruses isnot affected by this inactivation process. The batch is processed into avaccine as described in Examples 1-3.

Example 9 A batch prepared as described in Example 8 is subjected tohydroxylamine inactivation during 25 hours at 37 C. After this time,inactivation is discontinued by the addition of 15 cc. of acetone. Afterthis hydroxylamine treatment the batch is found to be free from activeSV40 and active polioviruses. The antigenic activity of the poliovirusesis not affected. The batch is processed into a vaccine as described inExamples 1-3.

Example 10 To 450 cc. of the suspension of polioviruses used in Examples8 and 9 are added 50 cc. of a bimolar hydroxylamine solution; the Wholeis thoroughly mixed and allowed to stand at 37 C. After 25 hours, theSV40 and the polioviruses are inactivated. The batch is then processedinto a vaccine as described in Examples 1-3.

We claim:

1. A process for the preparation of an inactivated viral suspensionadaptable to the manufacture of antipoliomyelitis vaccine, which processcomprises adding hydroxylamineto a suspension of poliomyelitis virusescontaining simian virus 40 at a temperature from 4 C. to 40 C. and at apH from 4.5 to 7 to give a concentration of hydroxylamine in saidsuspension from 0.01 to 5.0 mols per liter, and, after said suspensionis free of infectious viruses, removing unreacted hydroxylamine byaddition to the suspension of a carbonyl compound selected from thegroup consisting of acetone, acetylacetone, acetonylacetone, acetoaceticacid ethyl ester, and acetoacetic acid methyl ester.

2. A process as in claim 1 wherein oximes formed by the reaction of saidhydroxylamine and said carbonyl compound are removed from saidsuspension.

After i I 3. A process as in claim 2 wherein said oximes are soluble andare removed by dialysis.

4. A process as in claim 2 wherein said oximes are insoluble and areremoved by centrifugation.

5. A process as in claim 2 wherein said oximes are insoluble and areremoved by filtration.

6. A process as in claim 1 wherein a member selected from the groupconsisting of aluminum hydroxide and aluminum phosphate is added to saidsuspension.

7. A process as in claim 1 wherein the amount of carbonyl compound addedto said suspension is from 1 to 10 times the stoichiornetric amountrequired to react with the added hydroxylamine.

8. A process as in claim 1 wherein said temperature is about 20 C. andsaid pH is from about 4.5 to about 6.5.

9. A process as in claim 1 wherein said temperature is about 37 C. andsaid pH is about 7.

10. A process as in claim 1 wherein said concentration of hydroxylamineis about 0.2 mol per liter.

References Cited by the Examiner UNlTED STATES PATENTS 5/57 McLean167-78 6 OTHER REFERENCES Chem. Abstracts, 43: 3974g3975a (1949).

Chem. Abstracts, 46: 5180i-5181a; (1952).

Chem. Abstracts, 48: 10895d (1954).

Chem. Abstracts, 51: 137112 (1957).

Chem. Abstracts, 52: 6490g (1958).

Chem. Abstracts, 53: P 2271412 (1959).

Chem. Abstracts, 54: 5824i-5825a (1960).

Chem. Abstracts, 55: 95696 (1961).

Chem. Abstracts, 57: 1373d; 2673c; 6426i-2427a; p. 15573i (1962).

Gard The Virus of PoliomyelitisPhysical and Chemical Aspects, WorldHealth Organization, Monograph Series No. 26, pp. 215-235, 1955.

Holland et al., Enteroviral Ribonucleic Acid. Biological, Physical andChemical Studies, J. Exp. Med. 112(5), pp. 8214564, Nov. 1960.

Wallis et al., Stabilization of Poliovirus by Cations- CationicInactivation of vacuolating Virus (SV in Poliovirus Suspensions, TexasReports of Biology and Medicine, 19(3), pp. 683-705, fall 1961.

LEWIS GOTTS, Primary Examiner.

1. A PROCESS FOR THE PREPARATION OF AN INACTIVE VIRAL SUSPENSIONADAPTABLE TO THE MANUFACTURE OF ANTIPOLIOMYELITIS VACCINE, WHICH PROCESSCOMPRISES ADDING HYDROXYLAMINE TO A SUSPENSION OF POLIOMYELITIS VIRUSESCONTAINING SIMIAN VIRUS 40 AT A TEMPERATURE FROM 4*C. TO 40*C. AND AT APH FROM 4.5 TO 7 TO GIVE A CONCENTRATION OF HYDROXYLAMINE IN SAIDSUSPENSION FORM 0.01 TO 5.0 MOLS PER LITER, AND AFTER SAID SUSPENSION ISFREE OF INFECTIOUS VIRUSES, REMOVING UNREACTED HYDROXYLAMINE BY ADDITIONTO THE SUSPENSION OF A CARBONYL COMPOUND SELECTED FROM THE GROUPCONSISTING OF ACETONE, ACETYLACETONE, ACETONYLACETONE, ACETOACETIC ACIDETHYL ESTER, AND ACETOCETIC ACID METHYL ESTER.